CN108530552B - Preparation of laminarin and its application in the preparation of antitumor drugs - Google Patents
Preparation of laminarin and its application in the preparation of antitumor drugs Download PDFInfo
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- CN108530552B CN108530552B CN201810383468.1A CN201810383468A CN108530552B CN 108530552 B CN108530552 B CN 108530552B CN 201810383468 A CN201810383468 A CN 201810383468A CN 108530552 B CN108530552 B CN 108530552B
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- C—CHEMISTRY; METALLURGY
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Abstract
昆布多糖的制备及在制备抗肿瘤药物中的应用，属于植物活性成分的药物医疗和保健应用领域。依次采用纯水提取、醇沉、Sevag法脱蛋白、透析分离纯化，然后浓缩冷冻干燥。昆布多糖中多糖含量为≥93.9％，且昆布多糖中不含有蛋白质；肿瘤包括Hela(人宫颈癌细胞)，SH‑SY5Y(人神经母细胞瘤细胞)，MCF‑7(人乳腺癌细胞)，U266(人骨髓瘤细胞)、RAW264.7(小鼠腹腔巨噬细胞细胞)等。本发明将昆布多糖用于抗癌药物的制备，以供癌症的治疗使用。The preparation of laminarin and its application in the preparation of antitumor drugs belong to the field of medicinal medical treatment and health care application of plant active ingredients. Extraction with pure water, alcohol precipitation, deproteinization by Sevag method, separation and purification by dialysis, and then concentration and freeze-drying were performed in sequence. The polysaccharide content in laminarin is ≥93.9%, and laminarin does not contain protein; tumors include Hela (human cervical cancer cells), SH‑SY5Y (human neuroblastoma cells), MCF‑7 (human breast cancer cells), U266 (human myeloma cells), RAW264.7 (mouse peritoneal macrophage cells) and the like. In the present invention, laminarin is used for the preparation of anticancer drugs for the treatment of cancer.
Description
Technical Field
The invention relates to an anti-tumor effect of laminarin, belonging to the field of medical treatment and health care application of active ingredients of marine algae.
Background
Leukemia (Leukemia), also known as Leukemia, is a malignant tumor of the hematopoietic system. The etiology of the disease is the malfunction of hematopoietic tissues in the bone marrow due to mutations in intracellular deoxyribonucleotides. Stem cells in the bone marrow can be produced as thousands of red blood cells and white blood cells per day. Patients with leukemia overproduce immature leukocytes, impairing other work of the bone marrow, which results in a reduced function of the bone marrow to produce other blood cells. Leukemia can spread to lymph nodes, spleen, liver, central nervous system and other organs.
Polysaccharides (also known as polysaccharides) generally refer to macromolecular compounds formed by linking more than one monosaccharide through glycosidic bonds, such as cellulose, hemicellulose, gums, glycogen, and the like. The polysaccharide has physiological activities of regulating immunity, resisting tumor, lowering blood sugar, reducing blood lipid, resisting radiation, resisting virus, resisting inflammation, resisting fatigue, resisting aging, etc.
Kelp (also known as kelp) is a group of cryptogamic plants living in the sea, mainly containing polysaccharides, natural proteins, cellulose, fats, minerals, nucleic acids, etc., and is one of the daily-life food materials in countries such as china, japan, india, korea, etc. Modern pharmacological research shows that the kelp contains brown algae, brown algae starch, fucoidan and the like, and plays an important role in aspects. Therefore, the isolation and search of polysaccharides having excellent physiological activities from laminaria japonica has become a focus of pre-research by experts and scholars at home and abroad.
Disclosure of Invention
The invention aims to provide a preparation method of laminarin and a new application of laminarin in preparing antitumor drugs.
The technical scheme of the invention is as follows: extracting polysaccharide from thallus laminariae, and allowing the extracted polysaccharide to act on tumor cells such as Hela (human cervical cancer cells), SH-SY5Y (human neuroblastoma cells), MCF-7 (human breast cancer cells), U266 (human myeloma cells), RAW264.7 (mouse abdominal cavity macrophage cells), etc.
In order to solve the technical problems, the invention provides a preparation method of laminarin and an application of the laminarin in tumor resistance.
The laminarin provided by the invention has certain anti-tumor activity through testing, can inhibit the growth of various tumor cells within the range of 0.043-1.4mg/mL by in vitro administration, and presents an obvious dose-effect relationship.
The invention relates to a preparation method of laminarin, which comprises the following steps:
1) extracting crude laminarin of kelp:
weighing dry thallus laminariae powder, adding buffer solution and enzyme at a certain ratio, performing enzymolysis at a certain temperature and pH for a period of time, and heating to 90 deg.C to inactivate enzyme; then adding a sodium carbonate solution for digestion; carrying out vacuum filtration, taking filtrate, precipitating laminarin in a colloidal state by hydrochloric acid, standing overnight, then slowly adding hydrochloric acid into the standing solution, adjusting the pH value to 1.0-2.0, and filtering; at normal temperature, adding a sodium carbonate solution into the filtered solid while stirring to dissolve the gel blocks until neutralization is finished; adding absolute ethyl alcohol into the neutralized solution to separate out a precipitate; filtering, and drying the precipitate in oven to obtain crude polysaccharide of thallus laminariae;
each 1.0g of thallus laminariae powder corresponds to 20-50ml of buffer solution; the above buffer solution may be: a buffer solution of disodium hydrogen phosphate-sodium dihydrogen phosphate-phosphoric acid, citric acid-sodium citrate, acetic acid-sodium acetate;
the enzyme is selected from: one or more of pectinase, cellulase and xylanase;
the mass concentration of the enzyme is as follows: 0.45-0.6 g/L;
the temperature during enzymolysis can be as follows: 35-55 ℃;
the pH value during enzymolysis is as follows: 5 to 6.5;
the enzymolysis time can be as follows: 2-4 h;
adding 2% sodium carbonate solution while stirring to dissolve the rubber block.
Adding 2 wt% sodium carbonate solution 40ml per 1.0g thallus laminariae powder, and digesting at 55 deg.C for 2.0 h. 2) Deproteinization by the protease-Sevag method:
weighing the crude kelp polysaccharide obtained in the step 1), dissolving the polysaccharide in pure water, adding papain, carrying out constant-temperature water bath at 40 ℃ for 2h, adding a freshly prepared Sevag solution, oscillating for 10min in a vortex and curdling oscillator, centrifuging, and absorbing an upper aqueous solution; the Sevag solution is a mixed solution of chloroform and n-butanol in a volume ratio of 4: 1.
Dissolving 0.5g of crude laminarin of the kelp into 200-300 ml of pure water with the temperature of 40 ℃, correspondingly adding 0.9ml of papain solution with the concentration of 0.1mg/ml, and correspondingly adding 20ml of freshly prepared Sevag solution after 2 hours of constant-temperature water bath with the temperature of 40 ℃.
3) And (3) dialysis separation and purification:
taking the supernatant obtained in the step 2), putting the supernatant into a dialysis bag, dialyzing the supernatant in distilled water for 72 hours, and continuously changing water in the middle; the cut-off molecular weight of the dialysis bag is more than 3500; and (4) freezing and vacuum-drying the concentrated solution obtained after dialysis at-60 ℃ to obtain laminarin.
4) And (3) column chromatography separation and purification:
performing column chromatography separation and purification by using Sephadex G-50 Sephadex as filler, eluting with pure water as mobile phase at flow rate of 0.5mL/min, checking the purity of the obtained fraction by high performance gel permeation chromatography, and freeze-drying at-60 deg.C under vacuum to obtain laminarin monomer.
Measuring laminarin content, analyzing and detecting the laminarin by using high performance gel permeation chromatography, wherein the result shows that the laminarin monomer purity is more than 82.3%; meanwhile, protein was determined to be negative by Coomassie brilliant blue G-250 method, and laminarin contained no protein.
Preparation of laminarin1H NMR, FIG. 1 of laminarin1H NMR spectrum shows that the hydrogen peaks of anomeric carbon region and sugar ring region are more, and the hydrogen assignment in purified laminarin is calculated according to integral area as follows: δ -5.097-5.154 (1H, -O-CH-),3.765-3.833(6H, -CH-),3.688(2H, -CH-),3.634(4H, -CH-)2-),3.501(1H,-O-CH-).
Infrared spectrum of laminarin, taking a proper amount of sample to be measured, tabletting with potassium bromide (KBr), placing on an IR detection table, and measuring at 4000-800 cm-1Scanning was performed to observe the peak profile, as shown in FIG. 2.
The IR spectrum of laminarin showed 3400cm-1、3290cm-1、1700cm-1、1418cm-1、1153cm-1And the like typical characteristic absorption peaks of polysaccharides; 850cm-1A characteristic absorption peak of alpha-glycosidic bond is nearby; 830cm-1The nearby absorption peak indicates that it contains a pyran ring.
Laminarin antioxidant activity:
measurement of DPPH radical scavenging ability
2.0mL of a DPPH ethanol solution (0.1mmol/mL) and 2.0mL of a laminarin solution (concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, 0.50, 0.80, and 1.0mg/mL, respectively) were put in a 10mL volumetric flask, and after stirring, the mixture was reacted at room temperature in the dark for 30min, and then the absorbance of the mixture at 517nm was measured.
Clearance SD (%) - [1- (a)i-Aj)/Ac]×100
Wherein: a. thec: 2mL of LDPPH plus 2mL of absolute ethanol
Aj: 2mL of absolute ethyl alcohol and 2mL of solution to be detected
Ai: 2mL of the solution to be detected in the presence of 2mL of the PPH
OH radical activity
0.1mL of 10mmol/L Fe3O40.1mL of a 10mmol/L ethanol solution of salicylic acid and 0.1mL of a laminarin solution (concentrations of 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, 0.50, 0.80 and 1.0mg/mL, respectively) were placed in a 96-well plate. Finally, 0.1mL of 8.8mmol/L H was added2O2The reaction was carried out at 37 ℃ for 0.5h, and the absorbance at each concentration was measured at 510nm with distilled water as a reference. Distilled water was used as a blank.
Clearance calculation formula: clearance (%) ═ a0-(A1-A2)]/A0×100
Wherein: a. the0As absorbance of a reference solution
A1Is absorbance after adding laminarin solution
A2Without adding a color-developing agent H2O2Absorbance of (A)
And (3) detecting the anti-tumor activity:
according to the dissolution of the sample, the sample was dissolved with sterile physiological saline and sterilized by filtration through a 0.22 μm filter membrane, and the concentrations of the laminarin sample and the positive drug were 10 mg/ml.
The selected tumor cells were incubated at 37 ℃ with 5% CO2And culturing in a complete culture medium containing 10% fetal calf serum under saturated humidity, plating when the cells grow logarithmically, adjusting the cell suspension concentration to 3000/ml, and inoculating in a 96-well culture plate with 180 μ l per well. After cells grow 12h in an adherent manner, 20 mul of detection samples (43.75, 87.5, 175, 350, 700 and 1400 mul/ml) and positive control drugs are added respectively, equal volume of physiological saline is added into a blank group at the same time, after the cells are incubated for 72h, cell supernatant is discarded, MTT solution with the final concentration of 0.5mg/ml is added, and the cells are cultured for 4h continuously. The supernatant was aspirated off, 150. mu.l DMSO was added to each well, the mixture was shaken on a shaker for 10min to dissolve the crystals sufficiently, and the absorbance was measured at 450nm using a microplate reader. Independent repetitionThree experiments were carried out.
Tumor cell growth inhibition (%) - (OD)Control-ODExperiment of)/(ODControl-ODBlank space)×100。
In the MTT experiment, the higher the inhibition rate, the stronger the action activity of the test substance.
The laminarin provided by the invention is mainly used for treatment and adjuvant therapy of leukemia, wherein tumors are not limited to leukemia, Hela (human cervical carcinoma cells), SH-SY5Y (human neuroblastoma cells), MCF-7 (human breast cancer cells), U266 (human myeloma cells), RAW264.7 (mouse abdominal cavity macrophage cells) and the like are also carried out, and the laminarin has strong killing effect on MCF-7 cell strains and is used for preparing anti-tumor or tumor treatment medicines.
The anti-tumor medicine comprises laminarin and a pharmaceutically acceptable carrier or conventional edible auxiliary materials, such as: starch, sucrose, lactose, powdered sugar, glucose, mannitol, etc.
The dosage form of the antitumor drug is any one pharmaceutically acceptable dosage form.
Compared with the prior art, the preparation method of the laminarin sample adopted by the invention has the following advantages:
1) greatly improves the purity of laminarin;
2) the natural laminarin is obtained by utilizing the marine product kelp, and the raw materials are sufficient and the cost is low; further improves the added value of the kelp product and realizes the high-value utilization of the raw materials;
3) compared with contrast drugs of 5-fluorouracil and paclitaxel, the laminarin of the invention has better effect of inhibiting the growth of tumors;
4) the thallus laminariae is used as traditional food material, and can be orally administered to enhance immunity and promote health.
Drawings
FIG. 1 preparation of laminarin1H NMR spectrum
FIG. 2 Infrared Spectrum of Laminarin
FIG. 3 DPPH radical scavenging efficiency of laminarin
FIG. 4 hydroxyl radical scavenging efficiency of laminarin
FIG. 5 comparison of cell growth inhibition rates of laminarin against Hela (human cervical carcinoma cells), SH-SY5Y (human neuroblastoma cells), MCF-7 (human breast carcinoma cells), U266 (human myeloma cells), RAW264.7 (mouse peritoneal macrophage cells) after 72h of action.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1: the preparation method of laminarin comprises the following steps:
1) extracting crude laminarin of kelp:
precisely weighing 1.0g of dry thallus laminariae fine powder, adding citric acid-sodium citrate buffer solution and 0.5g/L of pectinase according to the material-liquid ratio (1g:40ml), performing enzymolysis at 35 deg.C and pH of 5.5 for 3h, and heating to 90 deg.C to inactivate enzyme; then adding 40ml of 2% sodium carbonate solution, and digesting for 2.0h at 55 ℃; filtering under reduced pressure, taking filtrate, precipitating laminarin in a colloidal state by using 5% hydrochloric acid, standing overnight, slowly adding dilute hydrochloric acid into the standing solution, adjusting the pH value to be 1.0-2.0, and filtering; at normal temperature, adding 2% sodium carbonate solution while stirring to dissolve the rubber block until the pH value is 7.5 for neutralization; adding a certain amount of absolute ethyl alcohol into the neutralized solution to separate out a precipitate; filtering, and drying the precipitate in an oven at 40-50 ℃ to obtain the crude laminarin of kelp.
2) Deproteinization by the protease-Sevag method:
precisely weighing 0.5g of crude kelp polysaccharide obtained in the step 1), dissolving the polysaccharide in 200-300 ml of 40 ℃ pure water, adding 0.9ml of 0.1mg/ml papain solution, adding 20ml of freshly prepared Sevag solution after carrying out constant-temperature water bath at 40 ℃ for 2h, shaking for 10min in a vortex oscillation instrument, centrifuging on a centrifuge for 5min at 4000r/min, and carefully sucking the upper aqueous solution. 1.0ml of supernatant was taken and the protein and polysaccharide contents were determined.
3) And (3) dialysis separation and purification:
taking the supernatant obtained in the step 2), putting the supernatant into a dialysis bag, dialyzing the supernatant in distilled water for 72 hours, and continuously changing water in the middle; the cut-off molecular weight of the dialysis bag is more than 3500; and (4) freezing and vacuum-drying the concentrated solution obtained after dialysis at-60 ℃ to obtain crude laminarin II of the kelp.
4) And (3) column chromatography separation and purification:
performing column chromatography separation and purification on the crude laminarin II obtained in the step 3) by taking Sephadex G-50 Sephadex as a filler, eluting by taking pure water as a mobile phase at the flow rate of 0.5mL/min, checking the purity of the obtained fraction by adopting a high performance gel permeation chromatography, and performing freeze vacuum drying at the temperature of-60 ℃ to obtain the laminarin monomer.
Example 2: laminarin antitumor Activity assay
1) Cell lines: hela (human cervical carcinoma cells), SH-SY5Y (human neuroblastoma cells), MCF-7 (human breast cancer cells), U266 (human myeloma cells), RAW264.7 (mouse peritoneal macrophage cells).
2) A sample to be tested: preparing the extracted laminarin with complete culture medium to a final concentration of 1500 mug/mL; positive drug: 5-fluorouracil and paclitaxel, prepared in complete medium, with a final concentration of 100 μ g/mL.
3) MTT method
Tumor cells at 37 ℃ with 5% CO2And culturing in a complete culture medium containing 10% fetal calf serum under a saturated humidity environment, plating when the cells grow logarithmically, adjusting the cell concentration to 3000 cells/well, and inoculating 180 mu l of the cells completely in a 96-well culture plate. After cells grow 12h adherent, 20 μ l of detection sample and positive control drug are added, the final concentration is 10 μ g/ml, equal volume of normal saline is added into the blank group at the same time, after cells are incubated for 72h together, the cell supernatant is discarded, MTT solution with the final concentration of 0.5mg/ml is added, and the culture is continued for 4 h. The supernatant was aspirated off, 150. mu.l DMSO was added to each well, the mixture was shaken on a shaker for 10min to dissolve the crystals sufficiently, and the absorbance was measured at 570nm using a microplate reader. Three experiments were independently repeated.
Tumor cell growth inhibition (%) (OD control-OD)Experiment of)/(ODControl-ODBlank space)×100。
In the MTT experiment, the higher the inhibition rate, the stronger the action activity of the test substance.
The tumor activity of laminarin is detected by MTT, and the laminarin can obviously inhibit the growth of Hela (human cervical carcinoma cells), SH-SY5Y (human neuroblastoma cells), MCF-7 (human breast cancer cells), U266 (human myeloma cells) and RAW264.7 (mouse abdominal cavity macrophage cells), as shown in figure 5. Experiments show that the proliferation inhibition rate of each tumor cell is in obvious positive correlation with the increase of the concentration, and the cell has very strong cytotoxic activity on SH-SY5Y cells; while the inhibition of proliferation of other tumor cells is relatively weak.
Example 3: laminarin sample preparation
The preparation method of the laminarin capsule comprises the following steps: taking laminarin powder in example 1, adding 1-15% of starch or other pharmaceutically acceptable carriers or conventional edible auxiliary materials (such as sucrose, powdered sugar, xylitol, polyethylene glycol, tween-80, glycerol, sodium cellulose, dextrin, sodium thiosulfate, gelatin and other conventional auxiliary materials), wherein the later preparation process and equipment of the preparation belong to the conventional technology in the pharmaceutical field, the invention is not limited to this, so the details are not described herein, using 90% ethanol as a wetting agent to prepare a soft material, sieving with a 20-mesh sieve for granulation, drying at 60 ℃, sieving with a 20-mesh sieve for finishing to obtain qualified granules, and encapsulating to obtain 0.050g of laminarin/granules.
Claims (7)
1. An application of laminarin in preparing medicine for treating tumor is SH-SY 5Y;
the preparation method of laminarin comprises the following steps:
1) extracting crude laminarin of kelp:
weighing dry thallus laminariae powder, adding buffer solution and enzyme at a certain ratio, performing enzymolysis at a certain temperature and pH for a period of time, and heating to 90 deg.C to inactivate enzyme; then adding a sodium carbonate solution for digestion; carrying out vacuum filtration, taking filtrate, precipitating laminarin in a colloidal state by hydrochloric acid, standing overnight, then slowly adding hydrochloric acid into the standing solution, adjusting the pH value to 1.0-2.0, and filtering; at normal temperature, adding a sodium carbonate solution into the filtered solid while stirring to dissolve the gel blocks until neutralization is finished; adding absolute ethyl alcohol into the neutralized solution to separate out a precipitate; filtering, and drying the precipitate in oven to obtain crude polysaccharide of thallus laminariae;
2) deproteinization by the protease-Sevag method:
weighing the crude kelp polysaccharide obtained in the step 1), dissolving the polysaccharide in pure water, adding papain, carrying out constant-temperature water bath at 40 ℃ for 2h, adding a freshly prepared Sevag solution, oscillating for 10min in a vortex and curdling oscillator, centrifuging, and absorbing an upper aqueous solution; the Sevag solution is a mixed solution of chloroform and n-butanol in a volume ratio of 4: 1;
3) and (3) dialysis separation and purification:
taking the supernatant obtained in the step 2), putting the supernatant into a dialysis bag, dialyzing the supernatant in distilled water for 72 hours, and continuously changing water in the middle; the cut-off molecular weight of the dialysis bag is more than 3500; freeze vacuum drying the concentrated solution at-60 deg.C to obtain laminarin;
4) and (3) column chromatography separation and purification:
performing column chromatography separation and purification by using Sephadex G-50 Sephadex as filler, eluting with pure water as mobile phase at flow rate of 0.5mL/min, checking the purity of the obtained fraction by high performance gel permeation chromatography, and freeze-drying at-60 deg.C under vacuum to obtain laminarin monomer.
2. The use of laminarin as defined in claim 1 for the preparation of a medicament for the anti-or treatment of tumors, wherein step 1) comprises 20-50ml of buffer solution per 1.0g of laminaria powder; the buffer solution is selected from: disodium hydrogen phosphate-sodium dihydrogen phosphate-phosphoric acid, citric acid-sodium citrate, acetic acid-sodium acetate buffer solution.
3. The use of laminarin according to claim 1 for the preparation of a medicament for the anti-or treatment of tumors, wherein the step 1) enzyme is selected from the group consisting of: one or more of pectinase, cellulase and xylanase.
4. The use of laminarin according to claim 1 for the preparation of a medicament for the anti-or treatment of tumors, wherein the enzyme of step 1) is present in a concentration by mass of: 0.45-0.6 g/L; the temperature in the step 1) enzymolysis can be as follows: 35-55 ℃; the pH value during enzymolysis is as follows: 5 to 6.5; the enzymolysis time can be as follows: 2-4 h.
5. The use of laminarin according to claim 1 for the preparation of a medicament for the treatment or prevention of tumors, wherein step 1) is carried out by adding a 2% sodium carbonate solution while stirring to dissolve the gel mass.
6. The use of laminarin as defined in claim 1 for the preparation of a medicament for the anti-or treatment of tumors, wherein 40ml of 2 wt% sodium carbonate solution is added per 1.0g of laminaria powder in step 1) and digested at 55 ℃ for 2.0 h.
7. The use of laminarin according to claim 1 for the preparation of a medicament for the anti-tumor or treatment of tumor, wherein the step 2) comprises dissolving 0.5g of crude laminarin in 200-300 ml of 40 ℃ pure water, adding 0.9ml of 0.1mg/ml papain solution, and adding 20ml of freshly prepared Sevag solution after 2h in 40 ℃ constant temperature water bath.
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| CN112626150A (en) * | 2020-12-28 | 2021-04-09 | 南京泛成生物科技有限公司 | Extraction method and application of laminarin |
| CN114392283A (en) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | anti-IPNV kelp extract and application thereof |
| CN114392285A (en) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | Preparation and application of kelp extract for resisting IHNV and IPNV co-infection |
| CN114392284A (en) * | 2022-01-26 | 2022-04-26 | 中国水产科学研究院黑龙江水产研究所 | Preparation and application of anti-IHNV kelp extract |
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